Immunoglobulin (antibody) is mainly present in blood and body fluids and binds to microorganisms (e.g. bacteria and viruses) that have invaded the body or to the cells infected by microorganisms, while recognizing them as antigens. When an antibody has bound to an antigen, a phagocyte (e.g., leukocyte or macrophage) engulfs the antigen-antibody complex to remove them from the body, or an immunocyte (e.g., lymphocyte) binds to the complex to induce immune reactions. The immunoglobulin thus has an important role in the defense mechanism against infections.
The immunoglobulin may be separated by separation/purification from biocomponents (mainly blood), or separation/purification from the cells (e.g., hybridoma), for example. Since more than 100,000 kinds of microorganisms, proteins, DNA, RNA, and viruses exist in the biocomponent, it is necessary to separate such other constituents from the immunoglobulin.
Multiple immunoglobulin molecules may also form immunoglobulin aggregates (mainly dimers) by bonding to each other via non-covalent bonds. The immunoglobulin aggregate is considered to be a cause of the side effects that occur when an immunoglobulin preparation is injected intravenously into the human body, such as shock reaction (e.g., cyanosis or pressure drop), airway symptom (e.g., respiratory difficulty), or rash. The immunoglobulin tend to agglomerate to produce larger multimers which may cause cloudiness or precipitation in the immunoglobulin solution. The immunoglobulin may bond to another protein, a bacterium, a virus, or the like to form a large protein aggregate that is referred to as an antigen-antibody complex. It is difficult to decompose the immunoglobulin aggregate that has been formed. Therefore, various methods have been proposed to remove such aggregates from a solution.
Several purification methods have been reported to separate immunoglobulin monomers from a solution containing immunoglobulin monomers and immunoglobulin aggregates. Examples of such purification methods include ion-exchange chromatography, hydrophobic chromatography, gel chromatography, the chemical treatment method, the adsorption method, and the ultrafiltration membrane method.
Ion-exchange chromatography and hydrophobic chromatography are effective for separating substances that differ in charge or hydrophobicity to a large extent. However, substances that differ in charge or hydrophobicity to only a small extent (e.g., immunoglobulin monomers and an immunoglobulin dimer) cannot be sufficiently separated. Moreover, a large amount of eluent or salt is necessary which is another drawback of these methods.
Gel chromatography that utilizes the size separation principle is effective for separating immunoglobulin monomers and immunoglobulin dimers that differ in size. However, since a large amount of immunoglobulin cannot be processed by gel chromatography, gel chromatography is time-consuming and costly.
A large amount of immunoglobulin can be processed by a chemical treatment that adds chemical substances. However, it is necessary to subsequently remove the chemicals used for the treatment from the solution. Moreover, inactivation or denaturation of the immunoglobulin easily occurs due to the treatment so that the immunoglobulin permeability may decrease.
The adsorption method does not necessarily exhibit a high immunoglobulin dimer removal efficiency. Moreover, it is necessary to remove the adsorbent as in the chemical treatment method.
In recent years, the ultrafiltration membrane method that is robust and convenient and can process a large amount of proteins has attracted attention. Various reports have been made on the ultrafiltration membrane method. When separating two components by ultrafiltration, it is understood that the fractionation performance and the permeation amount are affected by the cake layer that is formed on the surface of the ultrafiltration membrane. The molecular weight cut-off of the ultrafiltration membrane and the concentration of the particles (e.g., proteins) that form the cake layer are speculated to be important factors for the formation of the cake layer. However, since it is difficult to analyze the cake layer, few methods have been available for controlling cake layer formation.
For example, Non-patent Document 1, which reports separation of bovine serum albumin and lysozyme, does not contain any description on a cake layer analysis or a control method. Non-patent Document 1 is also silent about separation of immunoglobulin monomers and dimers.
As an immunoglobulin membrane separation method, Patent Document 1 discloses a method that removes globulin dimers by filtration using an ultrafiltration membrane formed of a polysulfone polymer. However, since the method disclosed in Patent Document 1 is a dead-end method, a large amount of immunoglobulin is captured in the membrane so that a thick cake layer is formed (i.e., clogging of the membrane occurs). Therefore, the immunoglobulin monomer permeability is quite low (about 40%), Moreover, the filtration speed and the filtration capacity are also decreased. This impairs the industrial effectiveness of the method disclosed in Patent Document 1.
Patent Document 2 discloses a method that removes immunoglobulin aggregates by dead-end filtration using a regenerated cellulose hollow fiber membrane. However, since the pore diameter (molecular weight cut-off) of the membrane is large, the immunoglobulin dimer is removed to only a small extent. Therefore, the method disclosed in Patent Document 2 is not sufficient for separating the immunoglobulin dimer.
When a large amount of impurities are contained in a solution, a cross-flow filtration method that rarely causes clogging of the membrane is generally utilized taking account of the membrane capturing capacity. Patent Document 3 discloses a method that removes immunoglobulin dimers by cross-flow filtration using an ultrafiltration membrane. However, Patent Document 3 is silent about the immunoglobulin monomer/dimer fractionation performance. In Patent Document 3, since filtration is performed at a very low concentration, it is considered that a cake layer that can separate immunoglobulin monomers and dimers is not sufficiently formed.
Patent Document 4 discloses a method that separates biocomponents that differ in molecular weight by a factor of less than 10 by cross-flow filtration while maintaining a level within the range of 5 to 100% of the flux at the transition point. Patent Document 4 discloses separation of proteins having a molecular weight of 100,000, but discloses no data about immunoglobulin monomer/dimer separation.
Patent Document 5 discloses a method that removes aggregates from an aqueous solution of immunoglobulin fractionated from human plasma, and filters the solution using a polyolefin porous membrane in the presence of a stabilizer with surface activity to reduce the anti-complement activity. However, Patent Document 5 discloses no data about immunoglobulin monomer/dimer separation. Moreover, Patent Document 5 states that it is preferable to use a membrane having a pore diameter that allows immunoglobulin aggregates (trimer and higher structures) to pass through the membrane. Thus, it is difficult to separate immunoglobulin monomers and immunoglobulin dimers using such a membrane.
Patent Document 6 states that immunoglobulin dimers can be removed using a membrane for separating plasma from blood. However, it is considered that the removal effect of such a membrane does not depend on the relationship between the sizes of the dimer and the pore. Instead the removal effect of the membrane is given due to adsorption of immunoglobulin dimers on the surface of the membrane caused by interaction between the membrane material and immunoglobulin dimers. Therefore, it is necessary to use the membrane under conditions (e.g., the pH of the solution and the ion intensity) in which adsorption occurs. Often the desired removal effect may not be obtained due to only a small change in conditions. When using the above method, the solution is likely to be contaminated with microorganisms (e.g., bacteria and viruses) from the outside during the operation. It is very important to prevent such a situation when producing a medicine. Therefore, it is necessary to inactivate or remove such microorganisms contained in the immunoglobulin solution which has been subjected to immunoglobulin dimer-removal treatment.
Cross-flow filtration is generally performed by means of constant-volume filtration that adds a diluent to the biocomponent solution in an amount equal to that of the immunoglobulin permeation solution (see Patent Documents 3 and 4). However, since the concentration of the biocomponent solution decreases as filtration proceeds, it may take long time in order to achieve a recovery rate of 80% or more.
Aggregates may be produced or cloudiness may occur in the immunoglobulin solution during cross-flow filtration using an ultrafiltration membrane. It is considered that this occurs when the immunoglobulin itself or associated impurities are insolubilized and aggregate due to stress applied to the immunoglobulin as a result of ultrafiltration. Since aggregation or cloudiness affects the permeation amount and the fractionation performance, it is important to suppress aggregation or cloudiness.
Aggregation of the immunoglobulin may be suppressed by adjusting the salt concentration or controlling the pH of the solution (Patent Document 7). A phosphate is used as an aggregation inhibitor. However, since a sufficient aggregation inhibiting effect is not obtained when the salt concentration is low, the salt used as an aggregation inhibitor is normally added in excess. As a result, precipitation of the immunoglobulin may occur. In this case, a subsequent desalting process is necessary and this increases the cost. Also, the immunoglobulin may be adversely denaturated when suppressing aggregation by controlling the pH of the solution.
Patent Document 8 discloses a method for suppressing formation of aggregates and cloudiness using sorbitan. However, the permeability decreases when adding sorbitan in an amount necessary to achieve a sufficient suppression effect.
When purifying the immunoglobulin, virus clearance ability is required in each purification step. The Paul-Ehrlich Institute (Germany) recommends that the sum of the log reduction values (LRV), of purification methods that differ in principle, be 10 or more (enveloped virus) or 6 or more (non-enveloped virus). A membrane that separates immunoglobulin monomers is also required to exhibit virus clearance ability. However, the virus clearance ability of an ultrafiltration membrane that separates immunoglobulin monomers and immunoglobulin dimers has not been reported.    Patent Document 1: JP S62-3815 B    Patent Document 2: JP H6-279296 A    Patent Document 3: Japanese Patent No. 3746223    Patent Document 4: Japanese Patent No. 3828143    Patent Document 5: JP H7-78025 B    Patent Document 6: JP S61-69732 A    Patent Document 7: JP 2004-267830 A    Patent Document 8: WO02/013859    Non-Patent Document 1: Separation Science and Technology, 33(2), 169-185 (1998)